Stimuli-responsive supramolecular assemblies consisting of small molecules are attractive functional materials for biological applications such as drug delivery, medical diagnosis, enzyme immobilization, and tissue engineering. By use of their dynamic and reversible properties, many supramolecular assemblies responsive to a variety of biomolecules have been designed and synthesized. This review focuses on promising strategies for the construction of such dynamic supramolecular assemblies and their functions. While studies of biomolecule-responsive supramolecular assemblies have mainly been performed in vitro, it has recently been demonstrated that some of them can work in live cells. Supramolecular assemblies now open up new avenues in chemical biology and biofunctional materials.

Spatiotemporal control of fluidity inside a soft matrix by external stimuli allows real-time manipulation of nano/micromaterials. In this study, we report a two-photon-responsive peptide-based supramolecular hydrogel, the fluidity of which was dramatically controlled with high spatial resolution (10 μm×10 μm×10 μm). The off–on switching of the Brownian motion of nanobeads and chemotaxis of bacteria by two-photon excitation was successfully demonstrated.

Spatiotemporal control of fluidity inside a soft matrix by external stimuli allows real-time manipulation of nano/micromaterials. In this study, we report a two-photon-responsive peptide-based supramolecular hydrogel, the fluidity of which was dramatically controlled with high spatial resolution (10 μm×10 μm×10 μm). The off–on switching of the Brownian motion of nanobeads and chemotaxis of bacteria by two-photon excitation was successfully demonstrated.

The modification of proteins with synthetic probes is a powerful means of elucidating and engineering the functions of proteins both in vitro and in live cells or in vivo. Herein we review recent progress in chemistry-based protein modification methods and their application in protein engineering, with particular emphasis on the following four strategies: 1) the bioconjugation reactions of amino acids on the surfaces of natural proteins, mainly applied in test-tube settings; 2) the bioorthogonal reactions of proteins with non-natural functional groups; 3) the coupling of recognition and reactive sites using an enzyme or short peptide tag–probe pair for labeling natural amino acids; and 4) ligand-directed labeling chemistries for the selective labeling of endogenous proteins in living systems. Overall, these techniques represent a useful set of tools for application in chemical biology, with the methods 2–4 in particular being applicable to crude (living) habitats. Although still in its infancy, the use of organic chemistry for the manipulation of endogenous proteins, with subsequent applications in living systems, represents a worthy challenge for many chemists.

Specific turn-on detection of enzyme activities is of fundamental importance in drug discovery research, as well as medical diagnostics. Although magnetic resonance imaging (MRI) is one of the most powerful techniques for noninvasive visualization of enzyme activity, both in vivo and ex vivo, promising strategies for imaging specific enzymes with high contrast have been very limited to date. We report herein a novel signal-amplifiable self-assembling ¹⁹F NMR/MRI probe for turn-on detection and imaging of specific enzymatic activity. In NMR spectroscopy, these designed probes are “silent” when aggregated, but exhibit a disassembly driven turn-on signal change upon cleavage of the substrate part by the catalytic enzyme. Using these ¹⁹F probes, nanomolar levels of two different target enzymes, nitroreductase (NTR) and matrix metalloproteinase (MMP), could be detected and visualized by ¹⁹F NMR spectroscopy and MRI. Furthermore, we have succeeded in imaging the activity of endogenously secreted MMP in cultured media of tumor cells by ¹⁹F MRI, depending on the cell lines and the cellular conditions. These results clearly demonstrate that our turn-on ¹⁹F probes may serve as a screening platform for the activity of MMPs.

Die Modifizierung von Proteinen mit synthetischen Sonden kann in vielfältiger Weise zur Aufklärung und Beeinflussung von Proteinfunktionen in vitro, in lebenden Zellen oder in vivo genutzt werden. Wir stellen hier die jüngsten Fortschritte bei den Methoden zur chemischen Proteinmodifizierung und ihrer Anwendung beim Protein-Engineering vor, wobei wir uns auf vier Strategien konzentrieren: 1) Biokonjugationsreaktionen von Aminosäuren an der Oberfläche natürlicher Proteine, hauptsächlich im Reagenzglas; 2) bioorthogonale Reaktionen von Proteinen mit nichtnatürlichen reaktiven Markergruppen; 3) Kupplung von Erkennungs- und reaktiven Regionen unter Verwendung eines Paars aus Enzym oder kurzem Peptid mit einem Sondenmarker zur Markierung natürlicher Aminosäuren 4) ligandendirigierte chemische Reaktionen zur selektiven Markierung endogener Proteine in lebenden Systemen. Zusammengenommen bieten diese Techniken ein nützliches Repertoire von Methoden zur Anwendung in der chemischen Biologie, wobei die drei letztgenannten auf unveränderte (lebende) Systeme anwendbar sind. Die organische Chemie zur Manipulation endogener Proteine ist zwar noch in einer frühen Phase, doch die nachfolgend beschriebenen Anwendungen sind ein lohnendes Ziel für Chemiker.

Controlling the morphology of supramolecular nanostructures in response to external stimuli is an important challenge in the development of functional soft materials. Here we show that a morphological transformation from 2D nanosheets to a network of 1D nanofibers is triggered by heating, which induces molecular conversion of a bolaamphiphile to a hydrogelator by means of a retro-Diels–Alder reaction, thereby producing a new heat-set supramolecular hydrogel. We anticipate that our design will be a starting point for more sophisticated supramolecular systems that integrate the thermodynamics of molecular assembly and the kinetics of chemical reactions to create complex supramolecular nanostructures.

Supramolecular hydrogels constructed through molecular self-assembly of small molecules have unique stimuli-responsive properties; however, they are mechanically weak in general, relative to conventional polymer gels. Very recently, we developed a zwitterionic amino acid tethered amphiphilic molecule 1, which gave rise to a remarkably stiff hydrogel comparable with polymer-based agarose gel, retaining reversible thermal-responsive properties. In this study, we describe that rational accumulation of multiple and orthogonal noncovalent interactions in the supramolecular nanofibers of 1 played crucial roles not only in the mechanical reinforcement but also in the multistimuli responsiveness. That is, the zwitterionic amino acid moiety and the CC double bond unit of the hydrogelator 1 can function as a pH-responsive unit and a light-responsive unit, respectively. We also demonstrated that this stiff and multistimuli-responsive supramolecular hydrogel 1 is applied as a unique mold for 2D and 3D-patterning of various substances. More significantly, we succeeded in the fabrication of a collagen gel for spatial patterning, culturing, and differentiation of live cells by using hydrogel 1 molds equipped with 2D/3D microspace channels (100–200 μm in diameter).

A complementary recognition pair of a short-peptide tag and a small molecular probe is a versatile molecular tool for protein detection, handling, and purification, and so forth. In this manuscript, we report that the binuclear Ni^II-DpaTyr (DpaTyr=bis((dipicolylamino)methyl)tyrosine) complex serves as a strong binding probe for an oligo-aspartate tag tethered to a protein. Among various binuclear metal complexes of M-DpaTyr (M=Zn^II, Ni^II, Mn^II, Cu^II, Cd^II, Co^III, and Fe^III), we have found that Ni^II-DpaTyr (1-2Ni2+) displays a strong-binding affinity (apparent binding constant: K_app≈10⁵ M⁻¹) for an oligo-aspartate peptide under neutral aqueous conditions (50 mM HEPES, 100 mM NaCl, pH 7.2). Detailed isothermal-titration calorimetry (ITC) studies reveal that the tri-aspartate D3-tag (DDD) is an optimal sequence recognized by 1-2Ni2+ in a 1:1 binding stoichiometry. On the other hand, other metal complexes of DpaTyr, except for Ni^II- and Zn^II-DpaTyr, show a negligible binding affinity for the oligo-aspartate peptide. The binding affinity was greatly enhanced in the pair between the dimer of Ni^II-DpaTyr and the repeated D3 tag peptide (D3×2), such as DDDXXDDD, on the basis of the multivalent coordination interaction between them. Most notably, a remarkably high-binding affinity (K_app=2×10⁹ M⁻¹) was achieved between the Ni^II-DpaTyr dimer 4-4Ni2+ and the D3×2 tag peptide (DDDNGDDD). This affinity is ≈100-fold stronger than that observed in the binding pair of the Zn^II-DpaTyr (4-4Zn2+) and the D4×2 tag (DDDDGDDDD), a useful tag-probe pair previously reported by us. The recognition pair of the Ni^II-DpaTyr probe and the D3×2 tag can also work effectively on a protein surface, that is, 4-4Ni2+ is strongly bound to the FKBP12 protein tethered with the D3×2 tag (DDDNGDDD) with a large K_app value of 5×10⁸ M⁻¹. Taking advantage of the strong-binding affinity, this pair was successfully applied to the selective inactivation of the tag-fused β-galactosidase by using the chromophore-assisted light inactivation (CALI) technique under crude conditions, such as cell lysate.

The creation of novel bioanalytical tools for the detection and monitoring of a range of important target substances and biological events in vivo and in vitro is a great challenge in chemical biology and biotechnology. Protein-based fluorescent biosensors—integrated devices that convert a molecular-recognition event to a fluorescent signal—have recently emerged as a powerful tool. As the recognition units various proteins that can specifically recognize and bind a variety of molecules of biological significance with high affinity are employed. For the transducer, fluorescent proteins, such as green fluorescent protein (GFP) or synthetic fluorophores, are mostly adopted. Recent progress in protein engineering and organic synthesis allows us to manipulate proteins genetically and/or chemically, and a library of such protein scaffolds has been significantly expanded by genome projects. In this review, we briefly describe the recent progress of protein-based fluorescent biosensors on the basis of their platform and construction strategy, which are primarily divided into the genetically encoded fluorescent biosensors and chemically constructed biosensors.

From a library of glyco-lipid mimics with muconic amide as the spacer, we found that 1, a glyco-lipid that has N-acetyl glucosamine and methyl cyclohexyl groups as its hydrophilic head and hydrophobic tails, respectively, formed a stable hydrogel (0.05 wt %) through hierarchical self-assembly of the lipid molecules into supramolecular nanofibers. The formation of the supramolecular hydrogel was verified by rheological measurements, and the supramolecular nanofiber was characterized as the structural element by transmission electron microscopy and atomic force microscopy observations. Absorption and circular dichroism spectroscopic measurements revealed that the muconic amide moieties of 1 are arranged in a helical, stacked fashion in the self-assembled nanofibers. Moreover, we unexpectedly found that the homogeneous distribution of the supramolecular nanofibers of 1 was greatly facilitated by the addition of polystyrene nanobeads (100–500 nm in diameter), as evaluated by confocal laser scanning microscopic observations. It is interesting that the obtained supramolecular hybrid matrix can efficiently encapsulate and distribute live Jurkat cells in three dimensions under physiological conditions. This supramolecular hybrid matrix is intriguing as a unique biomaterial.

A new chemical method to site-specifically modify natural proteins without the need for genetic manipulation is described. Our strategy involves the affinity-labeling-based attachment of a unique reactive handle at the surface of the target protein, and the subsequent selective transformation of the reactive handle by a bioorthogonal reaction to introduce a variety of functional probes into the protein. To demonstrate this approach, we synthesized labeling reagents that contain: 1) a benzenesulfonamide ligand that directs specifically to bovine carbonic anhydrase II (bCA), 2) an electrophilic epoxide group for protein labeling, 3) an exchangeable hydrazone bond linking the ligand and the epoxide group, and 4) an iodophenyl or acetylene handle. By incubating the labeling reagent with bCA, the reactive handle was covalently attached at the surface of bCA through epoxide ring opening. Either after or before removing the ligand by a hydrazone/oxime-exhange reaction, which restores the enzymatic activity, the reactive handle incorporated could be derivatized by Suzuki coupling or Huisgen cycloaddition reactions. This method is also applicable to the target-specific multiple modification in a protein mixture. The availability of various (photo)affinity-labeling reagents and bioorthogonal reactions should extend the flexibility of this strategy for the site-selective incorporation of many functional molecules into proteins.

The artificial regulation of protein functions is essential for the realization of protein-based soft devices, because of their unique functions conducted within a nano-sized molecular space. We report that self-assembled nanomeshes comprising heat-responsive supramolecular hydrogel fibers can control the rotary motion of an enzyme-based biomotor (F₁-ATPase) in an on/off manner at the single-molecule level. Direct observation of the interaction of the supramolecular fibers with a microbead unit tethered to the F₁-ATPase and the clear threshold in the size of the bead required to stop ATPase rotation indicates that the bead was physically blocked so as to stop the rotary motion of ATPase. The temperature-induced formation and collapse of the supramolecular nanomesh can produce or destroy, respectively, the physical obstacle for ATPase so as to control the ATPase motion in an off/on manner. Furthermore, this switching of the F₁-ATPase motion could be spatially restricted by using a microheating device. The integration of biomolecules and hard materials, interfaced with intelligent soft materials such as supramolecular hydrogels, is promising for the development of novel semi-synthetic nano-biodevices.

In a focused library of glycolipid-based hydrogelators bearing fumaric amide as a trans–cis photoswitching module, several new photoresponsive supramolecular hydrogelators were discovered, the gel–sol/sol–gel transition of which was pseudo-reversibly induced by light. Studying the optimal hydrogel by NMR spectroscopy and various microscopy techniques showed that the trans–cis photoisomerization of the double bond of the fumaric amide unit effectively caused assembly or disassembly of the self-assembled supramolecular fibers to yield the macroscopic hydrogel or the corresponding sol, respectively. The entanglement of the supramolecular fibers produced nanomeshes, the void space of which was roughly evaluated to be 250 nm based on confocal laser scanning microscopy observations of the size-dependent Brownian motion of nanobeads embedded in the supramolecular hydrogel. It was clearly shown that such nanomeshes become a physical obstacle that captures submicro- to micrometer-sized substrates such as beads or bacteria. By exploiting the photoresponsive property of the supramolecular nanomeshes, we succeeded in off/on switching of bacterial movement and rotary motion of bead-tethered F₁-ATPase, a biomolecular motor protein, in the supramolecular hydrogel. Furthermore, by using the photolithographic technique, gel–sol photopatterning was successfully conducted to produce sol spots within the gel matrix. The fabricated gel–sol pattern not only allowed regulation of bacterial motility in a limited area, but also off/on switching of F₁-ATPase rotary motion at the single-molecule level. These results demonstrated that the photoresponsive supramolecular hydrogel and the resulting nanomeshes may provide unique biomaterials for the spatiotemporal manipulation of various biomolecules and live bacteria.